Gene therapy holds great promise for new treatments for many diseases. Despite great pre-clinical successes, few gene therapy treatments have been effective in patients. In this grant application, is proposed a new type of gene therapy system, namely morpholino-nanoparticles. Morpholinos are highly effective for suppressing gene expression and, notably, for suppressing microRNA function. The nanoparticles used will possess the following features: morpholinos attached using a dithiol bond that will be cleaved in endosomes;a polymer coating that can disrupt endosomes for morpholino release into the cytoplasm;PEG chains to allow a long circulation half-life;galactose targeting to hepatocytes;an iron core for MRI and TEM detection;and a fluorophore for fluorescence techniques. The iron oxide and fluorophore components of the nanoparticle allow for a theranostic<approach, where the nanoparticle performance can be evaluated using imaging techniques. Due to the long circulation half-life of these nanoparticles and the galactose targeting, the nanoparticles should localize in the hepatocytes of the liver in vivo. Two approaches for reducing cholesterol production will be attempted: 1) PCSK9 knockdown and 2) miR-122 suppression. Cholesterol levels are correlated with the risk of heart disease and therefore this morpholino-nanoparticle system would be a treatment for individuals with elevated levels of cholesterol. However, the morpholino-nanoparticle delivery technology developed under this grant could subsequently be applied for therapy of other aspects of heart disease such as hypertrophy, cardiomyopathy and stenosis, or to other diseases such as cancer. The candidate is highly experienced in the synthesis of multifunctional nanoparticles that act as targeted contrast agents for medical imaging. The purpose of this award is to train the candidate to develop and apply novel nanoparticles for gene therapy purposes. The K99 mentored phase of the award will take place under the guidance of Prof. Roger Hajjar and Prof. Zahi Fayad of Mount Sinai School of Medicine. The focus of the training will be on the development of nanoparticles for gene therapy and the techniques required for analysis of mRNA and microRNA knockdown, i.e. PCR, Western blotting and Northern blotting. This mentored phase will set the stage for the R00 independent phase where the nanoparticle development and in vitro testing will continue and in vivo trials will be initiated. The results of this work should lead to the establishment of a fruitful line of investigation for the candidate that will reap benefits for our understanding of disease and human health. PUBLIC HEALTH RELEVANCE: Heart disease is the number one killer in the USA, and also leads to poor quality of life for those who suffer from it. High cholesterol levels lead to a higher likelihood a heart attack. While the class of drugs known as statins has been successful for lowering the cholesterol levels of many patients, there are still large populations for whom cholesterol levels are too high. In this application are proposed new methods for reducing cholesterol levels where nanoparticles deliver drugs called morpholinos that will shut down cholesterol production. We will focus on developing these morpholino-nanoparticle systems and evaluating their potential to reduce cholesterol levels.